Biological regulatory systems require the specific organization of proteins into multi-component complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important concern using two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins, and the degree to which observed interactions are specific. This proposal outlines a strategy to create a novel dual bait system, designed to allow single-step screening of libraries for proteins which interact with protein 1 of interest fused to DNA-binding-domain A (LexA), but do not interact with protein 2 fused to DNA-binding-domain B (lambda cI). This will be a powerful technique to screen for false positives. The dual bait strategy also will allow for characterization of related family member proteins (wild type versus a mutated form of the same protein). This adaptation will add a second bait vector and a strain, which will have the appropriate auxotrophic markers for each bait to respond to for positive interactions. Each bait fusion protein will have a second unique reporter readout (GFP and beta-galactosidase) for positive protein/protein interactions. PROPOSED COMMERCIAL APPLICATION: Not available.